In the production of proteins from animal serum or tissue, there is the potential of contamination by infectious agents that may be present in the primary starting material. The problem is particularly complex when considering the hazards of Slow Viruses such as Bovine Spongiphorme Encephalopathy (BSE) and Creutzfeld-Jacob Syndrome (CJS) and, possibly, various unknown viruses.
Witness the recent tragic example of HIV infection of many people due to the consequences of blood transfusions. Much of this happened before the pathogenic agents of AIDS became well known and has now resulted in prescreening of donors and the development of procedures to eliminate the HIV virus during the processing of sera and proteins. The more knowledge of the nature of the pathogenic agent is available, the easier it is to develop a suitable procedure to destroy it.
Historically, infectious agents such as bacteria, have well established methods of control that involve different forms of sterilization (e.g. steam sterilization, dry sterilization, pasteurization, sterile filtration, ethylene oxide sterilization, radiation inactivation, etc.). With viruses, there are also established methods which involve lowering of the pH to 4.0 or below, or use of organic solvents in high concentrations. Extended periods of heating at 60.degree. C. also may be used. In addition, UV treatment, formaldehyde and specific antiviral agents have been employed. However, these techniques sometimes have adverse effects on the proteins being isolated.
For some years now, new and previously unknown species of pathogenic agents have appeared and have been reported in scientific publications. These have been referred to as prions. The structure of these prions has been the subject of intense investigation and different points of view have been expressed. Some scientists feel they are extremely small viruses, while most experts now feel that prions are actually infectious proteins without a DNA or RNA core. This is the first contradiction to the scientific theory that DNA/RNA is essential for the duplication of infectious agents. While there is no firm evidence on the exact structure of these prions, there are diseases that have been identified recently both in humans and animals, that appear to be attributable to prions. No successful therapeutic treatments have been developed and as a result these diseases are always fatal. Adding to the problem is the fact that the incubation period can be up to 30 years and this factor presents a major challenge to the scientists involved.
One of these diseases, BSE (Bovine Spongiphorme Encephalopathy), the mysterious English-origin cattle disease, is the focus of much attention. Another is Scrapie of sheep and goat which actually may be the source of the BSE disease.
In humans there are diseases such as Kuru (an illness occuring with the ritual cannibals in Papua, New Guinea), the Creutzfeld-Jacob Syndrome and the Gerstmann-Straeussler Syndrome. The occurrence of these exotic illnesses is still fortunately very low, probably occuring at 1:1,000.000 but there are striking similarities when compared to the Alzheimer-syndrome. However, BSE is now reported to have reached epidemic proportions in England and is caused by the use of rendered materials in cattle feed and can originate with Scrapie infected sheep. Dairy cattle, in particular are at the highest measurable risk. A tragically similar incidence has occurred with humans.
During the production of human growth hormone from human pituitary glands collected from cadavers, the pathogenic agent of Creutzfeld-Jacob Syndrome was introduced. Several cases have now been reported in patients treated with this growth hormone. The patients were predominantly children, whereas the disease normally attacks adults over 50 years of age.
These examples point out the potential danger of these new diseases and the difficulties in diagnosing and treating them effectively.
The prions have very little, if any, nucleic acid and the prion protein is encoded by the host gene and is later transformed into a pathogen. Prions do not produce immune responses and the absence of antibodies makes diagnosis even more difficult. Prions are extremely resistant to physical and chemical methods of destruction. High concentrations of mineral acids or bases and preferably at high temperatures, Alkaline hypochlorite in high concentration, and temperatures above 150.degree. C. can eliminate prions. Mowever, these conditions effectively destroy or inactivate any biological properties of the native proteins as well. In order to produce a biological material that guarantees the absence of prions, conventional viruses and other infectious agents, a combination of inactivation methods appears to be necessary and must be supported by a biological test method to show that any infectious agents present (or deliberately added as controls) are definitely destroyed. Screening of the primary material is not a possibility as there is no known method to detect prions easily. Similarly, collecting the primary materials from areas where the disease has not been detected is not satisfactory when one considers the possible incubation period of up to 30 years.
A biological test system has been developed, where the material to be tested is injected into the brains of mice and the mice are then observed for pathological symptoms for up to 2 years. This method is impractical as a screening method for starting materials or as a batch testing method in production control. It can be used as a validation method of a new manufacturing process where prions are deliberately added to the starting material. However, safety precautions must be observed and the test animals must be maintained and tested in special P3 laboratories.